Neutrophil Extracellular Traps are Involved in the Innate Immune Response to Infection with Leptospira.

NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden.

Association of Toll-like receptor 2 Arg753Gln and Toll-like receptor 1 Ile602Ser single-nucleotide polymorphisms with leptospirosis in an Argentine population.

Toll-like receptor 2 (TLR2), a member of the Toll-like receptor family, plays an important role in the recognition of and subsequent immune response activation against leptospirosis in humans. The genetic polymorphism in TLR2 of an arginine to glutamine substitution at residue 753 (Arg753Gln) has been associated with a negative influence on TLR2 function, which may, in turn, determine the innate host response to Leptospira spp. This bacterium signals through TLR2/TLR1 heterodimers in human cells. The aim of the present study was to investigate the Arg753Gln single-nucleotide polymorphism (SNP) of the TLR2 gene, and the isoleucine to serine transversion at position 602 (Ile602Ser) of the TLR1 gene (previously associated with Lyme disease), in leptospirosis patients compared to healthy controls, carrying out a retrospective case/control study. The TLR2 polymorphism adenine (A) allele was observed in 7.3% of leptospirosis patients but was not found in the control group, whereas the guanine (G) allele of the TLR1 polymorphism was found in 63.6% of patients and 41.6% of controls. Susceptibility to leptospirosis disease was increased 10.57-fold for carriers of the TLR2 G/A genotype (P=0.0493) and 3.85-fold for carriers of the TLR1 G/G genotype (P=0.0428). Furthermore, the risk of developing hepatic insufficiency and jaundice was increased 18.86- and 27.60-fold for TLR2 G/A carriers, respectively. Similarly, the risk of developing jaundice was increased 12.67-fold for TLR1 G allele carriers (G/G and T/G genotypes). In conclusion, the present data suggest that the TLR2 Arg753Gln and TLR1 Ile602Ser SNPs influence the risk of developing leptospirosis and its severity.

Decay-accelerating factor 1 deficiency exacerbates leptospiral-induced murine chronic nephritis and renal fibrosis.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira, which can colonize the proximal renal tubules and persist for long periods in the kidneys of infected hosts. Here, we characterized the infection of C57BL/6J wild-type and Daf1-/- mice, which have an enhanced host response, with a virulent Leptospira interrogans strain at 14 days post-infection, its persistence in the kidney, and its link to kidney fibrosis at 90 days post-infection. We found that Leptospira interrogans can induce acute moderate nephritis in wild-type mice and is able to persist in some animals, inducing fibrosis in the absence of mortality. In contrast, Daf1-/- mice showed acute mortality, with a higher bacterial burden. At the chronic stage, Daf1-/- mice showed greater inflammation and fibrosis than at 14 days post-infection and higher levels at all times than the wild-type counterpart. Compared with uninfected mice, infected wild-type mice showed higher levels of IL-4, IL-10 and IL-13, with similar levels of α-smooth muscle actin, galectin-3, TGF-ß1, IL-17, IFN-γ, and lower IL-12 levels at 90 days post-infection. In contrast, fibrosis in Daf1-/- mice was accompanied by high expression of α-smooth muscle actin, galectin-3, IL-10, IL-13, and IFN-γ, similar levels of TGF-ß1, IL-12, and IL-17 and lower IL-4 levels. This study demonstrates the link between Leptospira-induced murine chronic nephritis with renal fibrosis and shows a protective role of Daf1.

Characterization of LIC11207, a novel leptospiral protein that is recognized by human convalescent sera and prevents apoptosis of polymorphonuclear leukocytes.

We report the study of a predicted outer-membrane leptospiral protein encoded by the gene lic11207. This protein is conserved in several pathogenic leptospiral strains but is absent in the saprophyte Leptospira biflexa. This putative outer-membrane protein has a domain of unknown function (DUF) 1565 found in several phylogenetically diverse bacteria and in the archaeon Methanosarcina acetivorans. The gene was cloned and expressed in Escherichia coli BL21 (SI) strain using the expression vector pDEST17. The 34 kDa recombinant protein was tagged with N-terminal hexahistidine and purified by metal-charged chromatography. The purified protein was used to assess: reactivity with human convalescent sera; in vivo expression; ability to activate endothelial cells (EC); and ability to modulate the apoptosis of polymorphonuclear cells (PMNs). The LIC11207 coding sequence was identified in vivo in the hamster renal tubules during experimental infection with Leptospira interrogans. The rLIC11207 showed significant antigenicity against human convalescent sera when compared with sera from healthy donors. The recombinant protein did not alter the surface expression of E-selectin or intercellular adhesion molecule 1 (ICAM-1) in EC and failed to induce the release of von Willebrand factor (vWF). Interestingly, rLIC11207 delayed apoptosis of PMNs suggesting a possible role of this protein during the infection.

Role of inducible nitric oxide synthase in the pathogenesis of experimental leptospirosis.

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is a radical effector molecule of the innate immune system that can directly inhibit pathogen replication. In order to study subsequent iNOS kidney expression in experimental leptospirosis, Golden Syrian hamsters and C3H/HeJ mice were infected intraperitoneally with 10(2) or 10(7) virulent Leptospira interrogans serovar Copenhageni (LIC) strain Fiocruz L1-130. Results showed increased levels of iNOS mRNA and protein in kidneys of infected animals when compared to that in mock-infected animals. To get a deeper insight into the role of iNOS in experimental leptospirosis, both subject species were treated or not treated with 4-aminopyridine (4-AP, 0.3mg/kg), an iNOS inhibitor. Treatment of infected hamsters with 4-AP accelerated the mortality rate to 100% by one day and increased the mortality rate from 20 to 60% in mice at 14 days post-infection. In kidney tissues, 4-AP treatment increased the bacterial burden, as demonstrated through leptospiral DNA quantification by real-time PCR, and aggravated tubulointerstitial nephritis. In addition, iNOS inhibition reduced the specific humoral response against LIC when compared to that in untreated infected animals. According to these results, iNOS expression and the resulting NO have an important role in leptospirosis.

Human embryonic stem cells and derived contractile embryoid bodies are susceptible to Coxsakievirus B infection and respond to interferon Iß treatment.

We studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P<0.05). All cell lines were susceptible to Coxsackievirus serotypes B1-5 infection as shown by RT-PCR detection of viral RNA, immunofluorescence detection of viral protein and infectivity titration of cell culture supernatants resulting in cell death. Supernatants infectivity titers 24-48 h post-infection ranged from 105-106 plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iß significantly reduced viral replication and associated cell death during a 24-48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection.

Lp95, a novel leptospiral protein that binds extracellular matrix components and activates e-selectin on endothelial cells.

OBJECTIVES: The study of a predicted outer membrane leptospiral protein encoded by the gene LIC12690 in mediating the adhesion process. METHODS: The gene was cloned and expressed in Escherichia coli BL21 (SI) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and used to assess its ability to activate human umbilical vein endothelial cells (HUVECs). RESULTS: The recombinant leptospiral protein of 95kDa, named Lp95, activated E-selectin in a dose-dependent fashion but not the intercellular adhesion molecule 1 (ICAM-1). In addition, we show that pathogenic and non-pathogenic Leptospira are both capable to stimulate endothelium E-selectin and ICAM-1, but the pathogenic L. interrogans serovar Copenhageni strain promotes a statistically significant higher activation than the non-pathogenic L. biflexa serovar Patoc (P<0.01). The Lp95 was identified in vivo in the renal tubules of animal during experimental infection with L. interrogans. The whole Lp95 as well as its fragments, the C-terminal containing the domain of unknown function (DUF), the N-terminal and the central overlap regions bind laminin and fibronectin ECM molecules, being the binding stronger with the DUF containing fragment. CONCLUSION: This is the first leptospiral protein capable to mediate the adhesion to ECM components and the activation of HUVECS, thus suggesting its participation in the pathogenesis of Leptospira.